The Mouse Model of Infection with Citrobacter rodentium

Nicolas Bouladoux1, Oliver J. Harrison2, Yasmine Belkaid1

1 NIAID Microbiome Program, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, 2 Mucosal Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 19.15
DOI:  10.1002/cpim.34
Online Posting Date:  November, 2017
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Abstract

Citrobacter rodentium is a murine mucosal pathogen used as a model to elucidate the molecular and cellular pathogenesis of infection with two clinically important human gastrointestinal pathogens, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). C. rodentium infection provides an excellent model to study different aspects of host‐pathogen interaction in the gut, including intestinal inflammatory responses during bacteria‐induced colitis, mucosal healing and epithelial repair, the induction of mucosal immune responses, and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. This unit provides detailed protocols for growing this bacterium, infecting mice by intragastric inoculation, measuring bacterial loads in feces and organs, and monitoring intestinal pathology induced by infection. Additional protocols describe steps needed to create frozen stocks, establish a growth curve, perform ex vivo organ cultures, isolate immune cells from the large intestine, and measure immune response by flow cytometry. © 2017 by John Wiley & Sons, Inc.

Keywords: Citrobacter rodentium; intestinal infection; colitis; mouse model

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Citrobacter rodentium Inoculum
  • Alternate Protocol 1: Cultivation of Non‐Antibiotic‐Resistant Citrobacter rodentium
  • Support Protocol 1: Preparation of Citrobacter rodentium Bacterial Stocks
  • Support Protocol 2: Creating a Growth Curve for Liquid Culture of Citrobacter rodentium
  • Basic Protocol 2: Intragastric Infection of Mice
  • Basic Protocol 3: Organ Recovery and Bacterial Enumeration
  • Basic Protocol 4: Histological Assessment of Citrobacter rodentium–Mediated Intestinal Inflammation
  • Support Protocol 3: Ex Vivo Organ Cultures for Quantification of Secreted Cytokines
  • Support Protocol 4: Isolation of Immune Cells From the Large Intestine
  • Support Protocol 5: Immunophenotyping of Lymphoid Cells by Flow Cytometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Citrobacter rodentium Inoculum

  Materials
  • Frozen stock of nalidixic acid–resistant strain ICC169 of C. rodentium (see protocol 3)
  • Dry ice
  • LB broth and agar plates (see recipe) with 50 μg/ml nalidixic acid
  • Phosphate‐buffered saline (PBS), sterile (Corning Cellgro)
  • 10‐μl inoculation loop, sterile (VWR International)
  • 125‐ml polycarbonate Erlenmeyer flasks, sterile (VWR International)
  • 37°C shaking incubator
  • Spectrophotometer
  • Disposable polymethyl methacrylate spectrophotometer cuvettes (Brandtech Scientific)
  • 17 × 100–mm 14‐ml polystyrene round‐bottom culture tubes, sterile (Corning Falcon)

Alternate Protocol 1: Cultivation of Non‐Antibiotic‐Resistant Citrobacter rodentium

  Materials
  • Citrobacter rodentium: nalidixic acid–resistant strain ICC169 or non‐resistant strain DBS100 (ATCC 51459)
  • MacConkey agar plates (see recipe)
  • LB broth and agar plates (see recipe) with 50 μg/ml nalidixic acid
  • Glycerol, sterile (Sigma‐Aldrich)
  • 125‐ml polycarbonate Erlenmeyer flask, sterile (VWR International)
  • 10‐μl inoculation loop, sterile (VWR International)
  • Spectrophotometer
  • Disposable polymethyl methacrylate spectrophotometer cuvettes (Brandtech Scientific)
  • 50‐ml conical tube, sterile (e.g., Greiner)
  • 1.5‐ml cryovials, sterile (e.g., Corning)

Support Protocol 1: Preparation of Citrobacter rodentium Bacterial Stocks

  Materials
  • Frozen stock of nalidixic acid–resistant strain ICC169 or non‐resistant strain DBS100 of C. rodentium (see protocol 3)
  • MacConkey agar plates (see recipe)
  • LB broth and agar plates (see recipe) with 50 μg/ml nalidixic acid
  • 10‐μl inoculation loop, sterile (VWR International)
  • 125‐ml polycarbonate Erlenmeyer flasks, sterile (VWR International)
  • Spectrophotometer
  • Disposable polymethyl methacrylate spectrophotometer cuvettes (Brandtech Scientific)

Support Protocol 2: Creating a Growth Curve for Liquid Culture of Citrobacter rodentium

  Materials
  • 6‐ to 8‐week‐old sex‐matched mice
  • Bacterial inoculum (see protocol 1)
  • Phosphate‐buffered saline (PBS), sterile (Corning Cellgro)
  • Sterile reusable stainless animal feeding needle, 22‐G × 1 in., straight, with a 1.25‐mm‐diameter bulbous tip (Braintree Scientific)
  • 1‐ml syringe, sterile (Covidien Monoject)
  • Additional reagents and equipment for anesthesia (optional; see unit 1.4; Donovan & Brown, )

Basic Protocol 2: Intragastric Infection of Mice

  Materials
  • C. rodentium–infected sex‐matched mice (see protocol 5)
  • Phosphate‐buffered saline (PBS), sterile (Corning Cellgro)
  • LB agar plates (see recipe) with nalidixic acid or MacConkey agar plates (see recipe), depending on C. rodentium strain
  • 5% (v/v) Lysol disinfectant
  • 70% (v/v) ethanol in a spray bottle
  • 2‐ml Safe‐Lock microcentrifuge tubes, sterile (Eppendorf)
  • 2‐ and 4.5‐ml homogenization tubes containing 1.4‐mm ceramic beads, sterile (MP Biomedicals Lysing Matrix D)
  • Dissection cutting board, sterile
  • Sterile dissection instruments:
    • dissecting pins
    • dressing forceps (curved and narrow at the tip)
    • iris scissors (straight and curved)
    • tweezers (curved and thin at the tip)
  • 100 × 15–mm Petri dishes, sterile (VWR International)
  • FastPrep Tissue homogenizer (MP Biomedicals)
  • Additional reagents and equipment for euthanasia (see unit 1.8; Donovan & Brown, )

Basic Protocol 3: Organ Recovery and Bacterial Enumeration

  Additional Materials (also see protocol 6)
  • 10% (v/v) neutral‐buffered formalin (Sigma‐Aldrich)
  • Hematoxylin solution (Mayer's; Sigma‐Aldrich)
  • Eosin Y solution (Sigma‐Aldrich)
  • Whatman paper (GE Healthcare)
  • Tissue‐processing histology cassettes (VWR International)
  • Histology pencil (VWR International)
  • Additional reagents and equipment for paraffin embedding, sectioning, and hematoxylin/eosin staining (optional; unit 21.4; Hofman & Taylor, )

Basic Protocol 4: Histological Assessment of Citrobacter rodentium–Mediated Intestinal Inflammation

  Additional Materials (also see protocol 6)
  • Complete medium (see recipe)
  • 48‐well polystyrene plate, sterile (Corning Costar)
  • 1.5‐ml Safe‐Lock microcentrifuge tubes (Eppendorf)

Support Protocol 3: Ex Vivo Organ Cultures for Quantification of Secreted Cytokines

  Additional Materials (also see protocol 6)
  • Harvest medium (see recipe)
  • Strip medium (see recipe)
  • Shake medium (see recipe)
  • 37.5% Percoll (see recipe)
  • Complete medium (see recipe)
  • Digest medium (see recipe)
  • Fine sharp straight scissors with a blunt end on one point
  • 125‐ml glass Erlenmeyer flask (VWR International)
  • 3 × 12–mm magnetic stir bar, sterile (VWR International)
  • Multi‐position magnetic stir plate (VWR International)
  • 50‐ and 600‐ml glass beakers (VWR International)
  • 10‐cm‐diameter fine mesh stainless steel strainer (e.g., kitchen strainer)
  • 15‐ and 50‐ml polypropylene centrifuge tubes, sterile (Greiner)
  • 40‐ and 70‐μm cell strainers, sterile (Corning Falcon)
  • 1‐ml syringe, sterile (Covidien Monoject)
  • Additional reagents and equipment for counting cells ( appendix 3B; Strober, )

Support Protocol 4: Isolation of Immune Cells From the Large Intestine

  Materials
  • Single‐cell suspension of intraepithelial or lamina propria lymphocytes (see protocol 9)
  • Complete medium (see recipe)
  • Phorbol 12‐myristate 13‐acetate (PMA; Sigma‐Aldrich)
  • Ionomycin (Sigma‐Aldrich)
  • Brefeldin A (GolgiPlug; BD Biosciences)
  • Hanks’ balanced salt solution (HBSS), 4°C (Corning Cellgro)
  • Unlabeled and fluorochrome‐labeled antibodies (see Table 19.15.2)
  • Fluorescent fixable viability dye (Molecular Probes)
  • Fixation/permeabilization solution, 4°C (eBioscience or BD Biosciences)
  • Permeabilization solution, 4°C (eBioscience or BD Biosciences)
Table 9.5.2   MaterialsAntibodies Typically Used to Assess Intestinal Immune Response by Flow Cytometry Following C. rodentium Infection

Antigen Antibody clone Antigen Antibody clone
CD4 RM4‐5 CD335 (NKp46) 29A1.4
CD8α 53.6.7 F4/80 BM8
CD8β eBioH35‐17.2 Foxp3 FJK‐16s
CD11b M1/70 Gr1 RB6‐8C5
CD11c N418 IFN‐γ XMG1‐2
CD16/32 (FcBlock) 93 IL‐17A eBio17B7 or TC11‐18H10.1
CD19 6D5 or eBio1D3 IL‐22 1H8PWSR or IL22JOP
CD44 IM7 Ly6G 1A8
CD45 30‐F11 RORγ(t) B2D
CD45R (B220) RA3‐6B2 TCRβ H57‐597
CD90.2 (Thy1.2) 53‐2.1 γδTCR eBioGL3
CD127 (IL‐7R) A7R34 Ter119 TER‐119
CD196 (CCR6) 29‐2L17 TNF‐α MP6‐XT22

  • 96‐well round‐bottom microtiter plates
  • Multi‐channel pipetter
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Figures

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Literature Cited

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